Prevention of Viral Infections after T Cell Depleted Allogeneic Stem Cell Transplantation By Infusion of Multi-Antigen Specific T Cell Products

Abstract Opportunistic viral infections and relapse are major complications in patients after T cell depleted allogeneic stem cell transplantation (TCD alloSCT). Since the application of unmodified donor lymphocyte infusion (DLI) early after alloSCT results in a high risk of graft versus host disease (GVHD), infusion of selected populations of virus-specific donor T cells can be an effective approach to safely restore anti-viral immunity early after alloSCT. As part of the EU FP7 consortium T Control, in this phase I/II study the feasibility and safety of the generation and administration of selected populations of donor-derived T cells targeting multiple antigens (Ag) is assessed. The multi Ag-specific T cell products contained T cells targeting cytomegalovirus (CMV), Epstein Bar virus (EBV) and adenovirus (AdV) as well as T cells targeting tumor associated Ag (TAA) and minor histocompatibility Ag (MiHA) to boost the graft versus leukemia (GVL) reactivity. To assess efficacy, in-vivo appearance or expansion of Ag-specific T cells, and the effect on viral reactivations and/or disease relapse was evaluated for 20 weeks after infusion until regular DLI was applied. HLA-A*02+ patients treated for a hematological malignancy with an HLA-matched TCD alloSCT from a CMV and/or EBV seropositive donor were included in this study. 6-8 weeks after alloSCT, T-cells directed against HLA-A*02-restricted peptides of CMV, EBV and AdV, and the TAA NY-eso, WT-1, RHAMM, PRAME and proteinase 3 were isolated using the reversible streptamer-nanobead technology (Juno) by cliniMACS selection out of the naïve and/or memory T cell compartment from 2*10^9 donor PBMC. Depending on the HLA-typing of the patient/donor additional streptamers targeting viral peptides in A*01, A*24, B*07 or B*08 were added to the selection procedure. In case of patient/donor MiHA disparity in the GvL direction, the HLA-A*0201/HA-1h streptamer was also added. This procedure allows purification of T cells under GMP conditions in 1 day. 20 multi Ag-specific T cell products were generated of which 19 met the release criteria. These products consisted of 0.5-12*10^6 cells containing purities of 46,0-94,3% target Ag-specific CD8+ T cells within the T cell compartment. In all products CMV and/or EBVas well as AdV virus-specific memory T cells were isolated comprising 99% of the target Ag-specific CD8+ T cells, while the other 1% included the TAA and MiHA specificities or CMV specific T cells from seronegative donors. 17 products were administered without infusion-related complications or GVHD; 2 patients experienced GVHD before infusion and did not receive their product. Of the 14 patients evaluated at this stage, 13 completed the follow-up period until DLI and 1 patient died during follow-up. No product-related adverse events were reported. In all 5 CMV- patients and in 3/9 CMV+ patients no CMV reactivation and no expansion of CMV-specific T cells was observed. The other 6 CMV+ patients experienced CMV reactivations. In 2/6 patients who received the product from a CMV+ donor CMV-specific T cells were detected with tetramer analysis and CMV was cleared. From the other 4 reactivating patients with CMV- donors 3 had circulating CMV-specific T cells already at the moment of infusion, whereas in 1 patient CMV-specific T cells clearly expanded after infusion, resulting in viral clearance in all 4 patients. All 14 donors were EBV+. In 7 patients EBV reactivations were observed, which coincided in 2/7 patients with the appearance of EBV-specific T cells and subsequent clearance of the virus. In 4/7 patients EBV reactivation was cleared without clear expansion of EBV-specific T cells. However, 1 patient required treatment for an EBV-PTLD, although ultimately EBV-specific T cells appeared and EBV was cleared. In none of the patients AdV DNA loads were detected in the follow-up period, while in 1 patient expansion of AdV-specific T cells was observed. Expansion of TAA and MiHA-specific T cells could not be demonstrated in-vivo using tetramer staining. More sensitive techniques will be required to visualize these cells. 2 patients showed relapse of their malignancy before DLI infusion. In this clinical study, we have shown that the reversible streptamer technology based generation and adoptive transfer of donor-derived multi Ag-specific T cell products is feasible and safe and can be used as a strategy to prevent viral infections in the interval between TCD alloSCT and DLI. Disclosures Germeroth: Juno Therapeutics: Employment.

Download Full-text

Myeloid Chimerism Reflects Engraftment of Donor Hematopoiesis, Whereas T Cell Chimerism Reflects Survival and Expansion of Donor and Recipient Residual Mature T Cells Early After T Cell Depleted Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v114.22.4475.4475 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4475-4475

Author(s):

Jessica C. Harskamp ◽

Esther H.M. van Egmond ◽

Hans L. Vos ◽

Stijn J.M. Halkes ◽

Roel Willemze ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cd8 T Cells ◽

Nk Cell ◽

Median Frequency ◽

Cell Compartment ◽

Hematopoietic Lineage ◽

Chimerism Analysis

Abstract Abstract 4475 Allogeneic stem cell transplantation (alloSCT) is frequently complicated by life-threatening graft versus host disease (GVHD). Previous studies demonstrated that T cell depletion (TCD) of the graft significantly decreases the incidence and severity of GVHD, and is associated with a higher percentage of patients with mixed chimerism (MC). In most studies chimerism analysis is performed on the total bone marrow (BM) leukocyte fraction, and changes in chimerism are related to engraftment. In this study we investigated whether MC in the total BM leukocyte fraction truly reflects engraftment or if it is influenced by survival and expansion of donor and recipient residual mature T cells, and whether hematopoietic lineage specific chimerism analysis is therefore a better method to determine engraftment. It is likely that chimerism analysis of the stem cell compartment is best reflected in peripheral blood (PB) in those cells that are continuously produced and short lived, such as monocytes and granulocytes, and therefore PB myeloid chimerism primarily reflects engraftment. In contrast, previous studies have shown by T cell receptor excision circle analysis that T cell neogenesis is virtually absent in the first 6 months after alloSCT, and that predominantly memory T cells are present in PB and BM. Therefore, we hypothesize that MC of these long lived T cells merely reflects survival and expansion of recipient and donor residual T cells. Since the life span of B and NK cells is longer than myeloid cells, but shorter than T cells, we anticipate that in the first 6 months after alloSCT, B and NK cell chimerism reflects a combination of survival and neogenesis. To analyze these hypotheses we performed hematopoietic lineage specific chimerism analysis on PB cells of 22 patients (median age 52 years, range 23-73, 11 males) receiving a TCD alloSCT between June and November 2008 after a myeloablative (n=11) or non myeloablative conditioning regimen (n=11) for AML, ALL, high risk MDS, multiple myeloma, CML, CLL or NHL. At intervals of 6 weeks PB was collected, and monocytes, granulocytes, B and NK cells, CD4+ and CD8+ T cells were sorted. The total leukocyte fraction was obtained by erythrocyte lysis of BM. DNA was isolated to perform chimerism analysis using short tandem repeats - PCR. Our results show that in the BM leukocyte fraction 47% of the patients were MC at 3 months after alloSCT, with a median frequency of patient cells of 4%. However, of the patients with MC in the total leukocyte fraction, 67% was complete chimeric in the myeloid subsets and MC in the T cell compartment. In the PB myeloid subsets (monocytes and granulocytes) less than 28% of the patients were MC during the first 6 months after alloSCT with a median frequency of patient cells less than 5%. In the B and NK cell subsets, at most time points more patients were MC (7-43%) with higher frequencies of patient cells (2-14%) compared to the myeloid subsets. The CD4 and CD8 T cell subsets showed the highest frequencies of MC in numbers of patients (31-61%) as well as the highest MC frequencies of patient cells (13-80%). Phenotypic analysis of the T cell compartment showed that 98% of the CD4 and CD8 T cells were memory cells during the first 6 months after alloSCT. Preliminary data indicate that the median percentage of donor derived T cells increased during the first 6 months after alloSCT, correlating with development of mild GVHD, suggesting that T cell chimerism is influenced by immunogenic triggers. In conclusion, these results illustrate that for engraftment and neogenesis of donor hematopoiesis, myeloid chimerism analysis provides more accurate information than total BM leukocyte chimerism analysis, since the results are greatly influenced by T cell chimerism. Since almost all T cells were memory cells within the first 6 months after alloSCT, T cell chimerism analysis reflects survival and expansion of mature donor as well as recipient T cells, and can therefore not be used to measure engraftment. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Prophylactic Infusion of Multi-Antigen Specific T-Cell Products to Prevent Complications after T Cell Depleted Allogeneic Stem Cell Transplantation — a Phase I/II Study

Blood ◽

10.1182/blood-2018-99-111749 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 119-119

Author(s):

Marthe C.J. Roex ◽

Peter van Balen ◽

Lothar Germeroth ◽

Lois Hageman ◽

Esther van Egmond ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Phase I ◽

Mhc I ◽

Donor T Cells ◽

The Moment

Abstract Viral infections and disease relapses are major complications between T cell depleted allogeneic stem cell transplantation (TCD alloSCT) and donor lymphocyte infusion (DLI). The prophylactic infusion of selected donor T cells may be an effective method to restore anti-viral and anti-tumor immunity early after TCD alloSCT. In this phase I/II study, we aimed to prevent these complications by the infusion of donor-derived T cell products containing CD8+ T cells directed against cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV) antigens (Ag), tumor associated Ag (TAA) and minor histocompatibility Ag (MiHA) 6-8 weeks after TCD alloSCT. The feasibility of multi-Ag specific T cell product generation and safety of early administration were investigated. Furthermore, clinical outcome and immune reconstitution were assessed. HLA-A2+ patients treated for a hematological malignancy with a ≥9/10 HLA-matched TCD graft from a CMV+ and/or EBV+ (un)related donor were eligible for inclusion. Donor T cells directed against HLA-A2-restricted peptides of CMV, EBV, AdV, and TAA NY-ESO-1, WT-1, RHAMM, PRAME and proteinase 3 were simultaneously isolated under GMP conditions in 1 day, using MHC I-Streptamers and CliniMACS selection out of the naïve and memory T cell compartment from 2*109 PBMC. Depending on patient/donor HLA-typing, additional MHC I-Streptamers targeting viral peptides in HLA-A1, -A24, -B7, or -B8 were added to the procedure, as well as the HLA-A2/HA-1h MHC I-Streptamer in case of MiHA disparity in the GVL direction. The incidence of GVHD, mortality, disease relapses and viral reactivations was monitored until 6 months after alloSCT. In addition, follow-up samples were stained with tetramers to analyze in vivo reconstitution of target-Ag specific T cells. Products were generated for 27/28 included patients that showed stable engraftment after TCD alloSCT; 1 patient died early after alloSCT. All donors were EBV+, 14/27 donors were CMV+. 26/27 products were successfully generated and contained a median of 5.2*106 cells (range 0.4 - 26.5*106) with a median purity of target-Ag specific T cells within the CD3+ compartment of 80.4% (range 46.0-99.9). Target-Ag specific T cells consisted for 99% of virus-specific memory T cells, while the remaining 1% included TAA, MiHA and naïve virus-specific T cells. 24 patients received their product (median 58 days after alloSCT; range 51-107) without infusion related complications; in 2 patients the infusion was cancelled due to acute skin GVHD on the day of infusion. During follow-up, 1 patient experienced skin GVHD requiring immunosuppressive therapy. One patient died due to complications of a nephrotic syndrome, probably unrelated to product infusion. Four patients showed disease relapse and were treated accordingly; coinciding expansion of TAA- or MiHA-specific T cells was not observed. None of the patients experienced AdV reactivations; in 2 patients AdV-specific T cells expanded after infusion. Four patients had detectable EBV loads; in one of these patients the EBV reactivation progressed to EBV-PTLD and he was treated with rituximab followed by DLI. In 2 patients, the rise in EBV load led to expansion of EBV specific T cells in vivo. 4/5 CMV+ patients with a CMV- donor had ongoing CMV reactivations at the moment of product infusion, CMV specific T cells could already be detected just before product infusion and reactivations were cleared within 7-84 days. Of the 8 CMV+ patients with a CMV+ donor, 3 patients remained free of CMV reactivations, 4 had already a reactivation at the moment of product infusion and 1 patient developed a CMV reactivation during follow up. One patient had progressed to CMV pneumonia within 2 weeks after infusion, requiring ganciclovir. In all 5 CMV reactivating patients, T cells directed against 2-3 CMV-specific epitopes expanded significantly and viral loads declined. TCR sequencing of the CDR3 region illustrated in vivo persistence and expansion of target-Ag specific T cells present in the product. In conclusion, we have shown that MHC I-Streptamer isolation based generation and adoptive transfer of donor-derived multi Ag-specific T cell products is safe and feasible. Moreover, efficacy of the prophylactic infusion is illustrated by expansion of target-Ag specific T cells in patients coinciding viral reactivations and the prevention of viral complications in the majority of patients between TCD alloSCT and DLI. Disclosures Germeroth: Juno Therapeutics: Employment, Equity Ownership.

Download Full-text

New Interleukin-15 Superagonist (IL-15 SA) Combined with Donor Lymphocyte Infusion (DLI) Significantly Enhances Graft-Versus-Tumor Activity after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v124.21.1118.1118 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1118-1118

Author(s):

Cavan Bailey ◽

Michelle M Panis ◽

Cihangir Buyukgoz ◽

Tulin Budak-Alpdogan ◽

Neal Flomenberg ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Cd8 T Cells ◽

Tumor Burden ◽

Donor Lymphocyte Infusion ◽

Hematopoietic Stem

Abstract Donor lymphocyte infusion (DLI) has been successfully used clinically to augment the graft-versus-tumor (GVT) effect following hematopoietic stem cell transplantation (HSCT) in relapsed patients. However, improvements can still be made in enhancing anti-tumor activity, reducing graft-versus-host disease (GVHD) and decreasing complications from opportunistic infections. Our studies present clear evidence of increased tumor clearance via cytokine therapy in combination with DLI as a way to “boost” the infused cells function. Interleukin-15 (IL-15) is a potent cytokine that increases CD8+ T and NK cells number and function in normal mice and recipients of stem cell transplantation. Despite this, obstacles remain for use of IL-15 therapeutically, specifically its low potency and short in vivo half-life. To overcome this, a new IL-15 superagonist (IL-15 SA-(ALT-803)) has been developed with a longer half-life and increased potency. Administration of IL-15 SA to recipients of CFSE labeled T cells increases proliferation of CD8+T cells and IFN-γ and TNF-α secretion from CD8+T cells. We developed a murine DLI model by titrating the dose of infused T cells in a parent-F1 model, and then combined IL-15 SA administration with DLI in murine recipients of allogeneic HSCT. In this model, lethally irradiated CB6F1 (H2Kb/d) mice were transplanted with T- cell depleted bone marrow cells from C57BL6 mice (H2Kb). All recipients of HSCT were also co-injected A20 B-cell lymphoma cells transfected with a luciferase-producing gene, which allows bioluminescent imaging and tracking of tumor progress in vivo. Mice receiving DLI (2.5 X 105 T cells) with IL-15 SA injections given at 1μg/mouse on days 17 and 24 post-BMT show less tumor burden and increased overall survival (p = 0.04) and decreased tumor growth (p = 0.02) (Figure 1). The IL-15 SA treated group had a significantly less weight loss than the control group (p = 0.007). No GVHD symptoms were noted via weekly clinical scoring, highlighting both the efficacy and overall safety of the IL-15 SA therapy. Furthermore, we evaluated T- cell exhaustion markers on CD8+ T cells in surviving mice. We found increased programmed death-1 (PD-1) expression on T cells even when the tumor burden is cleared. Treatment with IL-15 SA reduced PD-1 expression on donor CD8+ T-cells in mice surviving more than 120 days post-transplant. We conclude that IL-15 SA enhances CD8+ T cell function by increasing cytokine secretion and proliferation of T cells whereas could also prevent T cell exhaustion. We suggest that IL-15 SA is a long-waited lymphoid growth factor and has the potential to use in combination with DLI for the treatment of recurrent disease after HSCT. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Early Reconstitution of Antibody Secreting Cells after Allogeneic Stem Cell Transplantation

Journal of Clinical Medicine ◽

10.3390/jcm11010270 ◽

2022 ◽

Vol 11 (1) ◽

pp. 270

Author(s):

Martina Hinterleitner ◽

Clemens Hinterleitner ◽

Elke Malenke ◽

Birgit Federmann ◽

Ursula Holzer ◽

...

Keyword(s):

Innate Immunity ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

B Cells ◽

Stem Cell Transplantation ◽

B Cell ◽

Cell Transplantation ◽

Antibody Secreting Cells

Immune cell reconstitution after stem cell transplantation is allocated over several stages. Whereas cells mediating innate immunity recover rapidly, adaptive immune cells, including T and B cells, recover slowly over several months. In this study we investigated kinetics and reconstitution of de novo B cell formation in patients receiving CD3 and CD19 depleted haploidentical stem cell transplantation with additional in vivo T cell depletion with monoclonal anti-CD3 antibody. This model enables a detailed in vivo evaluation of hierarchy and attribution of defined lymphocyte populations without skewing by mTOR- or NFAT-inhibitors. As expected CD3+ T cells and their subsets had delayed reconstitution (<100 cells/μL at day +90). Well defined CD19+ B lymphocytes of naïve and memory phenotype were detected at day +60. Remarkably, we observed a very early reconstitution of antibody-secreting cells (ASC) at day +14. These ASC carried the HLA-haplotype of the donor and secreted the isotypes IgM and IgA more prevalent than IgG. They correlated with a population of CD19− CD27− CD38low/+ CD138− cells. Of note, reconstitution of this ASC occurred without detectable circulating T cells and before increase of BAFF or other B cell stimulating factors. In summary, we describe a rapid reconstitution of peripheral blood ASC after CD3 and CD19 depleted haploidentical stem cell transplantation, far preceding detection of naïve and memory type B cells. Incidence before T cell reconstitution and spontaneous secretion of immunoglobulins allocate these early ASC to innate immunity, eventually maintaining natural antibody levels.

Download Full-text

T-cell receptor-α repertoire of CD8+ T cells following allogeneic stem cell transplantation using next-generation sequencing

Haematologica ◽

10.3324/haematol.2018.199802 ◽

2018 ◽

Vol 104 (3) ◽

pp. 622-631 ◽

Cited By ~ 9

Author(s):

Cornelia S. Link-Rachner ◽

Anne Eugster ◽

Elke Rücker-Braun ◽

Falk Heidenreich ◽

Uta Oelschlägel ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Next Generation Sequencing ◽

T Cell ◽

Stem Cell Transplantation ◽

T Cell Receptor ◽

Cd8 T Cells ◽

Allogeneic Stem Cell Transplantation ◽

Cell Receptor ◽

Generation Sequencing

Download Full-text

Adoptive Transfer of Adenovirus Specific T-Cells in Children with Systemic Adenovirus Infection after Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v106.11.80.80 ◽

2005 ◽

Vol 106 (11) ◽

pp. 80-80

Author(s):

Tobias F. Feuchtinger ◽

Susanne Matthes-Martin ◽

Celine Richard ◽

Thomas Lion ◽

Klaus Hamprecht ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Adoptive Transfer ◽

Cell Transfer ◽

New Treatment ◽

T Cell Transfer

Abstract Allogeneic stem cell transplantation (SCT) has become an increasing treatment option for a variety of malignant and non-malignant disease. During immune reconstitution the host is at significant risk for viral infections. Human adenovirus (HAdV) infection is especially in children an important and serious complication. Virus-specific T-cells are essential for the clearance of HAdV, since antiviral chemotherapy has been insufficient to date. We present a new treatment option using virus-specific donor T-cells for adoptive transfer of immunity to patients with systemic HAdV-infection. We isolated in 6 patients with systemic HAdV-infection after SCT virus-specific T-cells of the donor, according to INF-γ secretion after short in vitro stimulation with viral antigen, resulting in a combination of CD4+ and CD8+ T-cells. Between 5-50x103/kg T-cells were infused for adoptive transfer. For follow-up, the infection and the in-vivo expansion of infused T-cells were evaluated. Isolated cells showed high specificity and markedly reduced but residual alloreactivity in-vitro. In three of four evaluable patients the infused T-cells underwent an in-vivo expansion and in these three patients the viral load decreased in peripheral blood after adoptive T-cell transfer. In-vivo expansion of specific T-cells was dose-independent. T-cell infusion was well tolerated. One patient experienced GvHD°II of the skin after T-cell transfer. In conclusion specific T-cell immunotherapy as a new treatment approach for children was performed in 6 cases of systemic HAdV-infection after allogeneic SCT. Induction of a specific T-cell response through adoptive transfer has been shown feasible and effective to protect from HAdV-related complications.

Download Full-text

CTL Responses Against Tumor Associated Antigens Are Part of the Graft Versus Leukemia-Effect and Protect from Relapse after Allogeneic Hematopoetic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v110.11.3231.3231 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3231-3231

Author(s):

Markus Kapp ◽

Stefan Stevanovic ◽

Kerstin Fick ◽

Juergen Loeffler ◽

Sen Mui Tan ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Cd8 T Cells ◽

T Cell Responses ◽

Proteinase 3 ◽

Post Transplantation ◽

Cell Responses

Abstract The Graft-versus-Leukemia (GVL) effect following allogeneic hematopoetic stem cell transplantation (HSCT) is one of the most prominent examples showing the ability of the immune system to eliminate malignant diseases. This effect was a strictly clinically described phenomenon, but in the last years T-cell responses against tumor-associated antigens (TAA) could partly be set in correlation with clinical benefit. Previously, TAA such as WT1 and proteinase-3 have been proposed as the targets for T-cells to establish a GVL effect. Now, we examined in addition other TAA (MUC1 and HM1.24) as possible T-cell targets of GVL related immune responses. We have defined new peptide epitopes from the MUC1 and HM1.24 antigens by the reverse immunology approach to increase the number of patients who can be screened and to expand the repertoire of immunologic monitoring as well as therapeutic approaches. A total of 25 patients after allogeneic stem cell transplantation have been screened and we are able to detect T-cell responses to both the MUC1 and HM1.24 antigens on top of the WT1 and the proteinase-3 antigen. Interestingly, we could detect a significant relationship between relapse and the absence of a T-cell response to TAA: Only 1/10 patients (10%) with TAA-specific CTL relapsed in contrast to 8/15 patients (53.3%) without TAA-specific CTL responses (p < 0.05). Furthermore, we demonstrated MUC1 peptides presented by HLA A*6801, B*0702 and B*4402 to be specifically recognized by CD3+/CD8+ T-cells. In conclusion, CD8+ T-cell responses directed to TAA might contribute to the GVL effect and are not limited to WT1 and proteinase-3. These observations clearly highlight both the importance and the potential of immunotherapeutic approaches in allogeneic stem cell recipients. Figure 1: New defined HLA class I epitopes predicted by computer analysis are recognized by specific CTL in patients post allogeneic HSCT. IFN-γ staining of PBMC from, patient No. 17 (AML, CR), 672 days post transplantation (A), patient No. 8 (AML, CR), 1035 days post transplantation (B) Cells were stimulated with 10μg/ml of the indicated peptides. Gates were set on lymphocytes by forward/side scattering (R1) and on CD3+/CD8+ cells (R2). Percentage numbers show peptide-specific CD3+/CD8+ T-cells from all CD3+/CD8+ T-cells. Figure 1:. New defined HLA class I epitopes predicted by computer analysis are recognized by specific CTL in patients post allogeneic HSCT. . / IFN-γ staining of PBMC from, patient No. 17 (AML, CR), 672 days post transplantation (A), patient No. 8 (AML, CR), 1035 days post transplantation (B) Cells were stimulated with 10μg/ml of the indicated peptides. Gates were set on lymphocytes by forward/side scattering (R1) and on CD3+/CD8+ cells (R2). Percentage numbers show peptide-specific CD3+/CD8+ T-cells from all CD3+/CD8+ T-cells.

Download Full-text

Exhaustion of CMV Specific T Cells with Enhanced PD-1 Expression In Persistent Cytomegalovirus Infection After Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood.v116.21.3912.3912 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3912-3912 ◽

Cited By ~ 1

Author(s):

Tomonori Kato ◽

Tetsuya Nishida ◽

Miho Murase ◽

Makoto Murata ◽

Tomoki Naoe

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Cd8 T Cells ◽

Culture Media ◽

Cmv Infection ◽

Control Serum ◽

And Control

Abstract Abstract 3912 Cytomegalovirus (CMV) is one of the most common pathogens causing morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT), despite preemptive treatments employing antiviral drugs. Cytotoxic T cells are indispensable to control CMV infections. Chronic viral infections with human immunodeficiency virus or hepatitis C virus were shown to be associated with exhausted T cells with high expression of the inhibitory molecule programmed death 1 (PD-1). Recently, it has been reported that PD-1 up-regulation on CMV specific T cells was associated with CMV infection after renal and liver transplantation. PD-1 expression on CMV specific T cells after HSCT has not been well examined. We evaluated the involvements of exhausted CMV specific T cells characterized by high PD-1 expression in persistent CMV infection after allogeneic HSCT. Peripheral blood mononuclear cells (PBMC) and serum were obtained from an HLA-A*2402-positive patient who had received bone marrow transplantation from an HLA-A, B, C and DR matched unrelated donor. This patient failed to eliminate CMV for more than one year after transplantation despite intermittent administration of ganciclovir and foscarnet. Control PBMC and serum were obtained from an HLA-A*2402-positive healthy volunteer because the Japan Marrow Donor Program prohibits blood collection for research use from donor. All blood was collected with written informed consent. We at first analyzed frequencies of CMV-specific CD8+ T cells in patient and control PBMC by flow cytometer using QYDPVAALF/A*2402-specific tetramer and CD8 antibodies. QYDPVAALF is derived from CMV pp65 protein and presented by the HLA-A*2402 molecule. Tetramer stained cells were detected in the patient PBMC but control PBMC (0.11% versus undetectable). Patient and control PBMC were stimulated by a synthetic peptide QYDPVAALF in culture media containing IL-2 for 14 days, and stained with QYD/A*2402-specific tetramer. Remarkably, post-stimulated patient PBMC contained only 0.54% of tetramer stained CD8+ T cells, whereas a more dramatic increase (14.1%) in control PBMC. We analyzed frequencies of IFN-g secreting CD8+ T cells in PBMC after stimulation with a peptide pool covering the whole CMV pp65 protein for 4 hours. Less patient CD8+ T cells produced IFN-g, compared with the control CD8+ T cells (0.5% versus 1.1%) These data demonstrate dysfunction of CMV-specific CD8+ T cells in the patient with persistent CMV infection. To examine the mechanism of dysfunction of CMV-specific CD8+ T cells, we analyzed the expression of PD-1 on CMV-specific CD8+ T cells 14 days after stimulation with QYDPVAALF peptide. Multiparameter flow cytometry and tetramer assay exhibited higher expression of PD-1 on CMV-specific CD8+ T cells generated from patient PBMC, compared with CMV-specific CD8+ T cells generated from control PBMC. To find out whether the engagement of PD-1 to its ligand (PD-L1) leads to T cell exhaustion, we stimulated patient PBMC with QYDPVAALF peptide for 14 days in the presence or absence of anti PD-L1 antibody which blocks PD-1/PD-L1 inhibitory pathway. Blockade of PD-1/PD-L1 pathway resulted in 3.9-fold increase in patient CMV specific T cells. These findings demonstrate that PD-1 is associated with the exhaustion of CMV specific CD8+ T cells during persistent CMV infection in this patient. To examine the effect of patient serum on CMV specific CD8+ T cells, we stimulated patient PBMC with QYDPVAALF peptide for 14 days in culture media with patient or control serum. CMV specific CD8+ T cells increased 4-fold and 55-fold in the presence of patient and control serum, respectively. Patient serum led to higher PD-1 expression on CMV specific CD8+ T cells, compared with control serum (Fig). These findings suggest that patient serum may contain what regulates PD-1 expression level of exhausted T cells. Further investigations to identify factors regulating PD-1 expression in patient serum are in progress. The identification of the factors may provide new strategies to improve exhausted T cell function. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Attenuated Acute Graft-Versus-Host Disease Following Allogeneic Stem Cell Transplantation In the Absence of RORγt.

Blood ◽

10.1182/blood.v116.21.3742.3742 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3742-3742

Author(s):

LeShara M Fulton ◽

Michael J Carlson ◽

James Coghill ◽

Michelle L. West ◽

Angela Panoskaltisis-Mortari ◽

...

Keyword(s):

T Cells ◽

Transcription Factor ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Graft Versus Host Disease ◽

Allogeneic Stem Cell Transplantation ◽

Graft Versus Host ◽

Donor T Cells

Abstract Abstract 3742 CD4+ T helper (Th) cells play a critical role in the development of Graft-versus-Host Disease (GvHD). The relative contributions of particular Th subsets to GVHD pathogenesis, however, are incompletely understood. In order to clarify the contribution of the Th17 subset to GVHD induction, we made use of mice knocked out at the RORgt locus (RORgt−/−), a transcription factor crucial for Th17 polarization. Methods: Haplotype matched and complete MHC mismatched murine HSCT models were used. For the haploidentical model C57BL/6 (H-2b, B6) mice served as donors while C57BL/6 × DBA2 F1 (H-2bxd, B6D2) mice functioned as recipients. Effector T cells (Teffs) were isolated from the spleens of wild type (WT) B6 and RORgt knockout mice backcrossed 7–8 generations onto a B6 background. B6D2 mice were lethally irradiated with 900 rads on day -1 and injected intravenously with 4 × 106 Teffs from WT or RORgt−/− mice supplemented with 3 × 106 WT T cell depleted bone marrow cells (TCD BM) on day 0. For the completely MHC mismatched model, BALB/c mice (H-2d) were lethally irradiated with 800 rads on day -1 and administered 5 × 105 WT or RORgt−/− Teffs supplemented with 5 × 106 B6 TCD BM on day 0. Results: B6D2 mice that received RORgt−/− Teffs displayed significantly attenuated GvHD, recovering from weight loss by day +31 and demonstrating 100% survival on day +60. Conversely, mice that received WT Teffs showed intense disease progression with 100% mortality by day +31 (Figure A, p<0.0001 for survival comparison between WT and RORgt−/− recipients using Fisher's exact test). Similar results were seen using the completely MHC mismatched model, with superior overall survival noted in those animals receiving RORgt −/− Teffs (put in p value here). Recipients of RORgt −/− T cells demonstrated statistically significant decreased TNF in serum compared to WT recipients (Figure B, p=0.001 comparing WT and RORgt−/− recipients using student's t test). Interestingly, despite the decreased severity of GvHD, serum concentrations of IFN-g were increased in recipients transplanted with RORgt −/− T cells. Chimerism studies post-transplant revealed complete donor reconstitution in recipients of both RORgt−/− and WT Teffs. Donor Teffs isolated from recipient livers post-transplant consistently demonstrated an activated phenotype, with low L selectin and high CD25 expression. Conclusions: T cell expression of the Th17 transcription factor, RORgt, is critical for the development of lethal GvHD following allogeneic stem cell transplantation in both the haploidentical and MHC complete mismatch models. GvHD attenuation in the absence of RORgt is not the result of an inability for donor T cells to undergo activation or to engraft in vivo. Interestingly, the absence of RORgt from donor T cells led to enhanced IFN-g in serum. Thus, in vivo, the Th17 pathway is critical for the induction of GvHD. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Adoptive Immunotherapy with Tregs and Tcons Ensures Low TRM and a Low Incidence of Post Transplant Leukaemia Relapse After HLA Haploidentical Transplants for Acute Leukemia

Blood ◽

10.1182/blood.v118.21.154.154 ◽

2011 ◽

Vol 118 (21) ◽

pp. 154-154

Author(s):

Mauro Di Ianni ◽

Franca Falzetti ◽

Alessandra Carotti ◽

Adelmo Terenzi ◽

Loredana Ruggeri ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Adoptive Immunotherapy ◽

Nk Cell ◽

Haematological Toxicity ◽

Post Transplant ◽

Leukaemia Relapse

Abstract Abstract 154 In full haplotype mismatched (HLA-haploidentical) stem cell transplantation we showed adoptive transfer of freshly isolated donor CD4+CD25+ FoxP3+ T regulatory cells (Tregs) followed by donor T cells (Tcons) prevented acute and chronic GvHD without any post-transplant immunosuppression, promoted lymphoid reconstitution and improved immunity against opportunistic pathogens (Di Ianni et al., Blood 2011). The major drawback was the extra-haematological toxicity of the conditioning regimen which included TBI, thiotepa, fludarabine and cyclophosphamide. To reduce regimen related toxicity we replaced cyclophosphamide with alemtuzumab, given 22 days before the Treg infusion to prevent it from interfering with adoptive T cell immunotherapy (Fig 1). The graft consisted of immunoselected Tregs (median 2×106/kg; range 1.6–4.8; FoxP3+ cells 92% ± 8 SD;), CD34+ cells (median 9.1×106/kg; range 8.1–10.9) and Tcons (median 1×106/kg; range 0.5–3). No post-transplant prophylaxis against GvHD was given. Since May 2010 18 patients (median age 43 years, range 23–61) with high risk acute leukaemia (16 AML, 2 ALL) have been transplanted. All sustained full donor-type engraftment. Neutrophils reached 0.5×109/L at a median of 12 days (range 9–28 days). Platelets reached 20×109/L and 50×109/L at median of 12 and 15 days, respectively (range 10–36 days and 11–55 days). CD4+ and CD8+ peripheral blood counts reached, respectively, 50/μL medianly on days 36 (range 27 – 120 days) and 34 (range 15– 85); 100/μL medianly on days 55 (range 27 – 147 days) and 48 (range 27 – 114); 200/μL on days 62 (range 37 – 177 days) and 49 (range 28 – 147). We observed a rapid development of a wide T-cell repertoire with specific CD4+ and CD8+ T cells for opportunistic pathogen antigen such as Aspergillus, Candida, CMV, ADV, HSV, VZV, Toxoplasma. Treg immunotherapy did not compromise post-transplant generation of donor-vs-recipient alloreactive natural killer (NK) cell repertoires in patients who received transplants from NK alloreactive donors (Ruggeri et al., Science 2002). Three of 16 valuable patients developed acute GvHD. Two responded to a short course of immunosuppressive therapy and at present (288 and 360 days after transplant) are alive and well with very good immunological reconstitution. The 3rd patient died of infectious complications. Two other patients died of non-leukemic causes (1 fulminant hepatitis 17 days post-transplant, 1 pneumonia 14 days post-transplant). The incidence of TRM is 17% (3/18). As hoped, extra-haematological toxicity was mild. One AML patient, who received a transplant from a non-NK alloreactive donor, relapsed 77 days post-transplant. Fourteen of the 18 patients are alive and well at a minimum follow-up of 3 months. This study shows adoptive immunotherapy with freshly isolated, naturally occurring Tregs is a feasible option in HLA-haploidentical stem cell transplantation since alloantigen-specific Tregs were efficiently activated in vivo and controlled alloreactivity of at least 1×106/kg Tcons without clinically significant inhibition of general immunity. Moreover Treg infusion did not weaken the GvL effect. The incidence of post-transplant leukaemia relapse was surprisingly low as only 1 patient has relapsed to date and even in our previous series no patient who was transplanted in CR has relapsed at a median follow-up of 25 months. Infusion of high numbers of Tcons in the absence of post-transplant immunosuppression can be hypothesized to exert a GvL effect. In addition, in patients who were transplanted from NK alloreactive donors, preservation of alloreactive NK cell repertoires played a key role in reducing the incidence of relapse. Disclosures: No relevant conflicts of interest to declare.

Download Full-text